We recently developed a method for nanomachining in hard materials using a femtosecond pulsed laser (Joglekar et al., 2003, 2004), which we have recently extended to the rapid production of three-dimensional (3D) subsurface nanofluidic and microfluidic networks in glass substrates. The overall aim of this proposal is to apply this unique capability to fabricate extremely high density and increased functionality micro- and nanofluidic devices for biomedical diagnostics and biochemical analysis. To our knowledge, this is the first time that three-dimensional complexity, with precision on the order of ten nanometers, has been available for glass microfluidics. We hypothesize that a variety of novel bioanalysis and drug-delivery systems may be fabricated for applications in medical diagnostics, purification, sequencing, and proteomic analysis. The overall aim of this R21 proposal is to aggressively explore the limits of this nanomachining technique, methodically explore transport of biomolecules within nanochannels constructed in this manner, and develop at least one highly integrated system with compelling characterization data. The proposed plan of research is unique and potentially of very high impact in that it enables entirely new classes of micro- and nanofluidic devices that are no longer limited to either two-dimensional geometries or multiple layers of polymer materials. Specific Aims are 1: Determine the physical and practical limits of 3-D femtosecond machining for micro- and nanofluidics applications 2: Thoroughly explore flow and transport in 3D femtosecond laser-machined micro- and nanochannels 3: Employ 3D nanomachining to rapidly prototype high-density flow control components and integrated analysis devices. The overall goal of this aim is to create a compelling demonstration of the use of a flow- control toolbox for microfluidics in glass substrates that could be used for microarray, microseparations, or micro-drug delivery systems. [unreadable] [unreadable] [unreadable]